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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Jan - 29 Mar 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (no TA102 or E.coli. WP2 tester strain, only 2-Aminoanthracene was used as positive control with S9 mix).
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no TA102 or E.coli. WP2 tester strain, only 2-Aminoanthracene was used as positive control with S9 mix
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): T-4113
- Physical state: white crystals
- Analytical purity: 99%
- Storage condition of test material: room temperature

Method

Target gene:
his operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other:
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other:
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
preliminary assay: 10, 50, 100, 500, 1000, 5000 µg/plate (with and without metabolic activation)
experiment I: 10, 50, 100, 500, 1000, 5000 µg/plate (with and without metabolic activation)
experiment II: 5, 10, 50, 100, 500, 1000 µg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-AA (9-Aminoacridine; 50µg/plate, TA1537, -S9); NF (2-Nitrofluorene; 5µg/plate, TA1538, 98, -S9); SA (Sodium Azide, 1µg/plate, TA1535, 100, -S9); 2-AA (2-Aminoanthracene, 1µg/plate, TA1538, 98, 100, 2.5µg/plate, TA1535, 1537, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduced background lawn, reduced number of colonies; formation of pinpoint colonies

Evaluation criteria:
Positive: A test article is considered a mutagen when it produces a reproducible, dose-related increase in the number of revertants in one or more strains. This increase should occur for at least three dose levels.
Negative: A test article is considered a nonmutagen when no dose-related increase in the number of revertants is observed in at least two independent experiments. The highest dose level tested for nontoxic solid substances and pure liquids is usually 5000µg/plate. For toxic compounds only the highest dose level tested should show evidence of toxicity.
Inconclusive: When a test article cannot be identified clearly as a mutagen or a nonmutagen in the assay, the results are classified as inconclusive.
Statistics:
No statistical analysis was performed.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only for TA1537 without metabolic activation in the first experiment at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was seen from 500µg/plate and upwards, for 1000 and 5000 µg/plate it interfered with counting of colonies wherefore plates were handcounted at those concentrations

RANGE-FINDING/SCREENING STUDIES: TA100 was used to screen for toxicity induced by the test article, with (4% v/v) and without metabolic activation, since no toxicity was seen the main experiments were carried out with 10% v/v S9 mix for metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results of Experiment I

 with or without S9 mix  Test substance concentration  Mean number of revertant colonies per plate (average of 3 plates  ± SD)            
    [µg/plate]  Base-pair substitution type     Frameshift type      
     TA100  TA1535  TA98  TA1537  TA1538
 -  0  119 ± 15  17 ± 9  17 ± 5  6 ± 1  18 ± 3
 -  10  114 ± 1  16 ± 3  20 ± 5 7 ± 2  12 ± 3
 -  50  114 ± 14  10 ± 1  24 ± 4  4 ± 1  14 ± 3
 -  100  104 ± 6  15 ± 1  19 ± 4  6 ± 1  6 ± 1
 -  500 P  92 ± 17  14 ± 8  15 ± 2  5 ± 3  9 ± 5
 -  1000 P  103 ± 15  13 ± 4  17 ± 6  8 ± 3  15 ± 5
 -  5000 P  125 ± 13  11 ± 3  15 ± 7 N  5 ± 1 T  17 ± 6
 positive control, -S9  Name  9-AA  SA  NF  9-AA  NF
   Conc. [µg/plate]  1  1  5  50  5
   Mean number of colonies/plate  457 ± 30  448 ± 9  1107 ± 80  571 ± 215  1503 ± 105
             
     TA100  TA1535  TA98  TA1537  TA1538
   0  153 ± 3  13 ± 1  34 ± 5  8 ± 2  32 ± 4
   10  137 ± 11  11 ± 11  42 ± 6  8 ± 4  39 ± 4
   50  134 ± 8  16 ± 7  42 ± 2  8 ± 1  32 ± 4
   100  175 ± 16  14 ± 1  42 ± 8  7 ± 3  28 ± 3
   500 P  162 ± 16  16 ± 3  41 ± 10  7 ± 3  25 ± 2
   1000 P  159 ± 10  11 ± 3  34 ± 4 N  9 ± 6  27 ± 7
   5000 P  132 ± 14 N  9 ± 3 N  39 ± 11 N  10 ± 4 N  30 ± 9 N
 positive control, +S9  Name  2 -AA   2 -AA   2 -AA   2 -AA   2 -AA
   Conc. [µg/plate]  1  2.5  1  2.5  1
   Mean number of colonies/plate 489 ± 64  158 ± 45  200 ± 16  68 ± 12

 178 ± 5

P = Precipitated

N = Normal background lawn

T = Thinning of background bacterial lawn, indicating toxicity

9 -AA = 9 -Aminoacridine

SA = Sodium Azide

NF = 2 -Nitrofluorene

2 -AA = 2 -Aminoanthracene

Table 2: Results of Experiment II

 with or without S9 mix  Test substance concentration  Mean number of revertant colonies per plate (average of 3 plates ±SD)            
    [µg/plate]  Base-pair substitution type     Frameshift type      
     TA100  TA1535  TA98  TA1537  TA1538
 -  0  114 ± 21  10 ± 4  14 ± 1  6 ± 2  14 ± 6
 -  5  125 ± 30  20 ± 5  21 ± 11  10 ± 1  15 ± 1
 -  10  130 ± 10  17 ± 2  19 ± 6  7 ± 2  13 ± 3
 -  50  133 ± 7  14 ± 1  19 ± 3  7 ± 3  14 ± 1
 -  100  128 ± 22  15 ± 6  19 ± 4  7 ± 3  13 ± 5
 -  500 P  121 ± 6  12 ± 2  18 ± 7  8 ± 2  11 ± 2
 -  1000 P  110 ± 11  10 ± 3  14 ± 3  5 ± 3  11 ± 3
 positive control, -S9  Name  9-AA  SA  NF  9-AA  NF
   Conc. [µg/plate]  1  1  5  50  5
   Mean number of colonies/plate  344 ± 22  207 ± 9  944 ± 49  414 ± 81  1060 ± 53
             
     TA100  TA1535  TA98  TA1537  TA1538
   0  142 ± 6  9 ± 3  37 ± 6  8 ± 1  29 ± 8
   5  164 ± 15  12 ± 3  37 ± 2  7 ± 3  35 ± 4
   10  156 ± 22  8 ± 2  35 ± 5  8 ± 1  27 ± 1
   50  137 ± 6  7 ± 5  35 ± 7  11 ± 2  25 ± 5
   100  146 ± 6  9 ± 2  39 ± 1  6 ± 1  22 ± 5
   500 P  144 ± 7  10 ± 3  32 ± 4  9 ± 2  27 ± 7
   1000 P  115 ± 1  8 ± 6  28 ± 16  5 ± 1  27 ± 9
 positive control, +S9  Name  2 -AA   2 -AA   2 -AA   2 -AA   2 -AA
   Conc. [µg/plate]  1  2.5  1  2.5  1
   Mean number of colonies/plate  745 ± 56  123 ± 8  660 ± 14  173 ± 41

 623 ± 13

P = Precipitated

N = Normal background lawn

T = Thinning of background bacterial lawn, indicating toxicity

9 -AA = 9 -Aminoacridine

SA = Sodium Azide

NF = 2 -Nitrofluorene

2 -AA = 2 -Aminoanthracene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative